NGS DNA Fragmentation
https://europepmc.org/articles/pmc6443234
Enzymes and reagents
Lyophilized DNase I (deoxyribonuclease I from Bovine pancreas) was obtained from Sigma-Aldrich (St Louis, MO, USA) and dissolved in the storage buffer (50% glycerol, 100 mM NaCl, 0.2 mg/ml BSA, 1 mM EDTA, 0.2 mM DTT, 20 mM Tris-HCl, pH = 8.0) up to 1 mg/ml.
SD DNA polymerase (50 U/μl) and the reaction buffer were supplied by Bioron GmbH, (Ludwigshafen, Germany). E.coli BL21(DE3) gDNA was supplied by Evrogen JSC (Moscow, Russia). dNTPs were obtained from Bioline Limited (London, UK).
NEBNext dsDNA Fragmentase and the NEBNext Ultra II DNA Library Prep kit were supplied by New England Biolabs, Inc. (Ipswich, MA, USA). dsDNA Fragmentation Through Polymerization (FTP)
For fragmentation, 200 ng of gDNA of the E. coli strain BL21(DE3) were added to the following reaction mixture: 1X reaction buffer for SD polymerase (Bioron GmbH), 3.5 mM MgCl2, 0.25 mM dNTPs (each), DNase I 1 ng/μl, SD DNA polymerase 1.5 U/μl. The total volume of the reaction was 25 μl. The reaction mixture was completed at 4°C (wet ice). The fragmentation of gDNA was carried out by two-step incubation: 20 minutes at 30°C and then 20 minutes at 70°C. For incubation, we used a thermal cycler with a heated lid. The reaction was stopped by cooling down the mixture to 10°C. The mixture was diluted 1:1 with sterile water, and fragmented DNA was purified with SPRI beads. DNA fragmentation with NEBNext dsDNA Fragmentase
gDNA of the E. coli strain BL21(DE3) was digested using NEBNext dsDNA Fragmentase (New England Biolabs, Inc.) according to the manufacturer’s protocol. Briefly, 200 ng of gDNA were added to the following reaction mixture (total volume 25 μl):1X Fragmentase Reaction Buffer v2, 10 mM MgCl, and 1X dsDNA Fragmentase. The mixture was incubated at 37°C for 20 minutes. The digestion was stopped by adding EDTA up to 100 mM. The mixture was diluted 1:1 with sterile water and fragmented DNA was purified with SPRI beads. Preparation of NGS libraries
We prepared four NGS libraries from four different samples of Fragmentase-digested gDNA and four NGS libraries from four different samples of FTP-digested gDNA. NGS libraries were generated using NEBNext Ultra II DNA Library Prep kit (New England Biolabs, Inc.) according to the manufacturer’s instructions. The conventional procedure for Fragmentase-digested DNA included repair of DNA ends with the NEBNext Ultra II End Prep Enzyme Mix, addition of adapters to the DNA fragments using NEBNext Ultra II Ligation Master Mix, and amplification of the adaptor-ligated DNA fragments with the NEBNext Ultra II Q5 Master Mix. The input amount of each DNA sample was 200 ng. The library indexing and amplification were performed for 5 PCR cycles as described in the kit’s manual.
NGS libraries from FTP digested gDNA were constructed using the NEBNext Ultra II DNA Library Prep Kit procedure, excluding the DNA end repair stage. The input amount of each DNA sample was 200 ng. The library indexing and amplification were performed for 5 PCR cycles with the NEBNext Ultra II Q5 Master Mix.
After the amplification stage, all libraries were quantified with a Quant-iT PicoGreen dsDNA Assay Kit (Molecular Probes, Inc., Eugene, OR, USA) and with the Agilent 2200 TapeStation Instrument with a D1000 Tape System (Agilent Technologies, Waldbronn, Germany), pooled (500 ng of each), and purified with AMPure XP beads.
the Fragmentation Through Polymerization method is a novel, robust, and simple method of DNA fragmentation which is suitable for NGS. In comparison with Fragmentase, it provides very similar characteristics for NGS libraries. Potential disadvantages of FTP are associated with biases of the enzymes used in the method, such as non-random DNA fragmentation and mismatch errors. These characteristics of FTP were compared with the Fragmentase method. The experimental data demonstrate that FTP yields higher quality random fragmentations (Fig 3) and better coverage of GC reach contents (Fig 4B) than Fragmentase. Levels of mismatch errors are similar for both methods. FTP generates a greater number of unmappable reads than Fragmentase, but the difference is less than 1% of all reads in the library.
The main advantage of the FTP method lies in the simplification of NGS library preparation by eliminating the DNA end repair and A-tailing stage from the protocol. In the result, the work time of the procedure can be decreased from 180 minutes to 110 minutes (the repair/A-tailing stage takes 70 minutes according to the manual). Additionally, it can reduce the price of the library preparation. For example, the current price of the NEBNext Ultra II DNA Library Prep kit for 24 reactions is 535 Euros; the price of the NEBNext Ultra II End Repair/dA-Tailing Module for 24 reactions is 262 Euros. Thus, the elimination of this module from the kit can decrease the primary cost of NGS library preparation.